The neuronal glycine transporter 2 interacts with the PDZ domain protein syntenin-1

Abstract

The glycine transporter subtype 2 (GlyT2) is localized at glycinergic axon terminals where it mediates the re-uptake of glycine from the extracellular space. In this study, we used the yeast two-hybrid system to search for proteins that interact with the cytoplasmic carboxy terminal tail region of GlyT2. Screening of a rat brain cDNA library identified the PDZ domain protein syntenin-1 as an intracellular binding partner of GlyT2. In pull-down experiments, the interaction between GlyT2 and syntenin-1 was found to involve the C-terminal amino acid residues of GlyT2 and the PDZ2 domain of syntenin-1. Syntenin-1 is widely expressed in brain and co-localizes with GlyT2 in brainstem sections. Furthermore, syntenin-1 binds syntaxin 1A, which is known to regulate the plasma membrane insertion of GlyT2. Thus, syntenin-1 may be an in vivo binding partner of GlyT2 that regulates its trafficking and/or presynaptic localization in glycinergic neurons.

Introduction

The amino acid glycine serves as a principal neurotransmitter at many inhibitory synapses in spinal cord and brainstem (Betz, 1992) and, in addition, as coagonist of the N-methyl-d-aspartate (NMDA) subtype of ionotropic glutamate receptors (Kemp and Leeson, 1993). Termination of glycine neurotransmission is thought to involve the rapid uptake of released glycine into presynaptic terminals and surrounding glial cells. Specific plasma membrane transporters mediate this process. So far, two types of glycine transporters have been cloned, glycine transporter subtype 1 (GlyT1) and glycine transporter subtype 2 (GlyT2) (reviewed in Gomeza et al., 2003a, Lopez-Corcuera et al., 2001). Both proteins are members of the family of Na⁺/Cl⁻-dependent neurotransmitter transporters characterized by 12 transmembrane segments and cytoplasmic N- and C-terminal tail regions Nelson, 1998, Schloss et al., 1994.

In situ hybridization and immunohistochemistry have shown that GlyT1 is localized in glial cells throughout the central nervous system (CNS), whereas GlyT2 is found in neurons of the spinal cord and brainstem Adams et al., 1995, Jursky and Nelson, 1995, Luque et al., 1995, Zafra et al., 1995. The restricted expression of GlyT2 matches that of strychnine-sensitive inhibitory glycine receptors; thus, GlyT2 constitutes a specific marker of inhibitory glycinergic nerve terminals. Recent studies with knockout mice have shown that GlyT1 activity is essential for the removal of glycine from the synaptic cleft of inhibitory synapses, whereas GlyT2 is needed for efficient reloading of synaptic vesicles with glycine Gomeza et al., 2003b, Gomeza et al., 2003c. Consistent with this functional assignment, immunoelectron microscopy revealed an enrichment of GlyT2 in the presynaptic nerve terminal membrane surrounding the neurotransmitter release sites Mahendrasingam et al., 2003, Spike et al., 1997.

During the past decade, an increasing number of proteins containing PDZ (for postsynaptic density protein-95, discs large, zona occludens-1) domains has been shown to interact with the carboxy terminal regions of different membrane proteins Fanning and Anderson, 1999, Harris and Lim, 2001, Sheng and Sala, 2001 and suggested to regulate the synaptic distribution and functional properties of ion channels and neurotransmitter receptors. Recently, Torres et al. (2001) have identified a PDZ domain protein, PICK1, as a binding partner of the human dopamine transporter (DAT), and shown that this interaction modulates transporter function. PICK1 (for protein interacting with C kinase 1) is a protein kinase C (PKC) substrate, as is MacMARCKS (for macrophage myristoylated alanine-rich C kinase substrate), a plasma membrane-associated protein which interacts with the serotonin transporter (SERT) (Jess et al., 2002). Notably, different Na⁺/Cl⁻-dependent neurotransmitter transporters including GlyT1 are known to undergo acute down-regulation in response to PKC activation Corey et al., 1994, Sato et al., 1995. This down-regulation involves redistribution of the transporter proteins from the plasma membrane to intracellular compartments (Blakely and Bauman, 2000). Thus, transporter interacting proteins like PICK1 and MacMARCKS may be implicated in the regulation of both transporter function and trafficking. In addition, such proteins may be crucial for the nerve terminal localization of these membrane proteins, as demonstrated in case of PICK1 for the presynaptic metabotropic glutamate receptor 7 (Boudin et al., 2000).

Here, we used the yeast two-hybrid system to search for novel binding partners of GlyT2 and identified the PDZ protein syntenin-1, which had originally been identified as a syndecan binding protein (Grootjans et al., 1997), as a specifically interacting protein. Furthermore, we present evidence that syntenin-1 can interact with syntaxin 1A, a SNARE (for soluble NSF attachment protein receptor) complex component that has been implicated in the trafficking of neurotransmitter transporters including GlyT2 (Geerlings et al., 2001). Our data are consistent with a role of syntenin-1 in GlyT2 trafficking and/or presynaptic localization.